The present invention relates to the cell regulation factor TTO 20 and DNA therefor, and its preparation and use for screening purposes for the discovery of modulators for TTO 20 activity.
The many biological effects of interleukin-1 (IL-1) include the action of IL-1 on the metabolism of many types of cells from the connective tissue. An example of cells of this type is articular chondrocytes. IL-1 inhibits the synthesis of proteoglycans (PG) by chondrocytes and stimulates the production of prostaglandin E2 and metalloproteinases, which are capable of degrading molecules of the extracellular matrix.
On the basis of experimental results, and the discovery of IL-1, PG fragments, and proteolytic enzymes in inflammation-modified joints, it was concluded that IL-1 plays a part in cartilage degradation in osteoarthrosis and rheumatoid arthritis (Beuton H P and Tyler J A, 1988, Biochem. Biophys. Res. Comm. 154:421-428; Aydelotte M B et al., Comm. Tiss. Res. 28:143-159; Wood D D et al., Arthritis Rheum. 26:975-983; Lohmander L S et al., Trans. Orthop. Res. Soc. 17:273). Matrix metalloproteases are potential candidates for starting points for a therapy with active compounds that interact with these enzymes. Until now, no actual molecular starting points have been identified that relate to early steps in the complex process that leads to cartilage degradation. For this reason, various approaches have been chosen in order to obtain molecular starting points of this type for a medicinal therapy of osteoarthrosis and rheumatoid arthritis.
Such an approach is described in European Patent Application EP 0 705 842 A2. The question was whether it would be possible to obtain potential molecular starting points for a medicinal therapy of IL-1xcex2-induced cartilage degradation on the RNA plane in human, articular chondrocytes from osteoarthritic cartilage.
For this purpose, genes were identified that are expressed differentially in diseased cartilage. Total RNA from IL-1xcex2 stimulated and nonstimulated human chondrocytes was subjected to a differential display of mRNA by reverse transcription and the polymerase chain reaction (DDRT-PCR). This method can be used for the identification and isolation of genes that are expressed differentially in two cell populations (Liang P and Gardee A B (1992), Science 257: 967-971; Liang P et al., A B (1993), Nucl. Acids Res. 21: 3269-3275; Bauer D et al. (1993), Nucl. Acids Res. 21:4272-4280). The key element of this technology is the use of a set of oligonucleotide primers, one of which binds to the polyadenylated tail of the mRNA, and the others are random decamers that bind to various other sites of the mRNA. Such mRNA subpopulations, which are defined by a specific set of primers, are amplified after reverse transcription and separated on DNA sequencing gels. Band patterns are seen that are characteristic for one of each of the cell lines studied. Thus, for example, 100 different primer combinations should afford 10,000 different PCR products, which represent at least approximately half of all the genes expressed in a cell line. A comparison of the band patterns of two different cell lines indicates those bands that correspond to differentially expressed genes. On the basis of this information, it is now possible to extract, to reamplify, to subclone, and to sequence bands of differentially expressed gene products from the gel.
However, this is to be qualified by saying that this method has a number of difficulties:
1. As a result of the high sensitivity of the DDRT-PCR, slightly artificial bands can result.
2. The analysis of complex gene expression patterns is difficult.
3. Only tiny amounts of RNA are available as starting material.
These difficulties cause uncertainty in the results obtained.
In European Patent Application EP 0 705 842 A2, a number of short DNA sequences are disclosed that have been identified in the manner described above. An analysis of these sequences showed that some are complete or have very great identity with the sequences of already known genes. Thus, a cDNA fragment having 100% identity with human osteopontin, another cDNA fragment having 97.2% identity with human calnexin, and a further fragment having 99.5% identity with human TNF-30 stimulated gene 6 (TSG-6) were found. Most of the fragments found, however, could not be assigned to any known gene based on the sequence corresponding to the fragment. This group of cDNA fragments also included the 400 bp-long clone TTO 20/2(2), 152 bp of which has been deciphered.
In the context of the present invention, the clone TTO 20/2 has now been investigated more closely. An antisense experiment is one method for investigating the functional meaning of the corresponding gene or gene product.
The expression of antisense RNA in human chondrosarcoma cells, which are regarded as model cells for cartilage differentiation, yielded indications of a role of TTO 20/2. The antisense approach is based on transforming the cultured cells with a vector that expresses antisense mRNA to TTO 20, but at the same time, the vector also expresses an indicator protein whose activity indicates whether antisense RNA was formed. Vectors of this type are called bicistronic or dicistronic vectors. The starting vector for the present constructs was pED4, whose construction has been described by Kaufmann et al. (Kaufmann et al., (1991), Nucl. Acids Res. 19: 4485-4490).
In antisense technology, the formation of a functional protein is restricted or even prevented via the expression of a complementary RNA (antisense RNA) that binds to the protein-encoding mRNA (sense RNA). In particular, as the present examples confirm, antisense RNA can be employed for subregions of the encoding mRNA, and for the 3xe2x80x2 or 5xe2x80x2 untranslated region, in order to prevent the formation of the target protein. With the aid of the vector, EST fragments that have no defined open reading frame can thus be used in order to work out the action of the protein that is finally encoded by the associated gene because the xe2x80x9cantisense-expressedxe2x80x9d EST switches off or decreases the reading of the encoding sense mRNA. If the synthesis of the target protein that is blocked in this way plays an important role in the cell, this has direct or indirect effects on cell division, cell growth, synthesis of regulated and expressed proteins etc. If the antisense expression prevents, for example, the formation of a factor that plays a role in signal cascades, then that cascade is disturbed.
If the factor is a transcription factor, the expression of a number of genes is disturbed. This can be recognized, for example, from morphologically visible changes that can be attributed to altered expression of secreted proteins, particularly proteases.
The genes, or products thereof, identified in this way can be employed as therapeutic targets for the search for pharmacologically active substances. Likewise, cells transformed in this way can be used in screening systems attempting to block the action of the antisense RNA.
DNA chip technology allows the direct analysis of such changes because the transcript profiles of transformed cells can be compared with untransformed or mock-transformed cells. The comparison then allows conclusions to be drawn as to whether an EST plays a crucial role in the context of a clinical picture and is thus suitable as a screening target. The use of the vector is thus also suitable for the discovery of novel targets and for the profiling of novel medicaments. The vector is particularly suitable for the synthesis of HTS systems for target validation. Expediently, EST clones are cultured in HTS formats, such as 96-well microtiter plates, and the insert DNA is amplified by means of PCR using suitable PCR primers that, for example, generate a PST cleavage site on the 3xe2x80x2 side and an Eco RI cleavage site on the 5xe2x80x2 side for cloning in one of the pED4 derivatives described. In a second step, the PCR fragments generated in this way are cleaved using Pstl and Eco RI and ligated into the dicistronic EGFP vector. The screening format here is retained. The ligated construct containing a PCR fragment is pipetted onto previously prepared eukaryotic cells, using commercially obtainable pipetting robots, along with suitable transfection agents, such as CaPO4, Fugene 6 (manufacturer: Boehringer, Mannheim; lipofectamine, Life Technologies, Eggenstein) or others, and the cells are transformed according to customary processes. Here too, the screening format is retained. The cells are incubated in the presence of CO2 according to customary processes and tested for fluorescence emission after 24-72 hours under automated conditions in a fluorescence scanner. Wells with transformed, fluorescent cells are then evaluated for changes in growth and changes in cell morphology, etc., as compared with transformed control cells that were transfected with the vector lacking a Pstl-Eco RI insert. If changes of this type occur, this indicates an essential action of the expressed antisense RNA. The isolation of the cloned DNA and the subsequent sequence analysis give an indication of the nucleotide sequence and thus the gene or the coded protein involved. In this way, the first functional indications of gene activities can be found whose function cannot itself be derived by means of the EST.
The invention therefore relates to the TTO 20 polypeptide comprising the amino acid sequence according to Table 1, SEQ. ID. NO.:10.
A further object of the invention is a substantially purified TTO 20 polypeptide, wherein said polypeptide is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ. ID. NO. 9 or degenerate variants thereof.
A further object of the invention is a substantially purified TTO 20 polypeptide, wherein said polypeptide is encoded by a polynucleotide that is at least 80% identical to the nucleic acid sequence of SEQ. ID. NO. 9 or degenerate variants thereof, preferably 85% identical to the nucleic acid sequence of SEQ. ID. NO. 9, more preferably 90% identical to the nucleic acid sequence of SEQ. ID. NO. 9 , and most preferably 95% identical to the nucleic acid sequence of SEQ. ID. NO. 9.
Another object of the invention is an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO.: 9, preferably base pair 1 to base pair 1305.
A further object of the invention is a polynucleotide having a nucleic acid that is at least 80% identical to the nucleotide sequence of SEQ. ID. NO. 9 or degenerate variants thereof, preferably 85% identical to the nucleic acid sequence of SEQ. ID. NO. 9, more preferably 90% identical to the nucleic acid sequence of SEQ. ID. NO. 9, and most preferably 95% identical to the nucleic acid sequence of SEQ. ID. NO. 9.
Another object of the invention is an antibody that recognizes the polypeptide of TTO20 comprising the amino acid sequence according to Table 1, SEQ. ID. NO.:10.
Another object of the invention is an expression vector construct comprising the polynucleotide of SEQ ID NO 9 and a host cell transfected or transformed with the expression vector construct.
Another object of the invention is a process for the preparation of the TTO 20 polypeptide comprising the steps of:
(a) culturing a host cell under conditions that result in the expression of said TTO 020 polypeptide; and
(b) isolation of said TTO 20 polypeptide from said host cell or culture medium.
A further object of the invention is a TTO 20 polypeptide prepared by this process.
Another object of the invention is a process for the identification of cell lines, cells, or tissues that express the TTO 20 polypeptide, wherein a nucleic acid probe is used to hybridize an RNA fragment derived from SEQ ID NO 9 in a biological sample.
A further object of the invention is a method for determining the complete polynucleotide sequence of the TTO20 polypeptide comprising the use of hybridization and/or PCR processes.
A further object of the invention is a method comprising the determination of the three-dimensional structure of the TTO 20 polypeptide and using said three-dimensional structure to design inhibitors or activators of said TTO 20 polypeptide.
A further object of the invention is a method for determining substances that influence the activity of TTO20 comprising an assay that measures the amount of TTO 20 polypeptide.
Another object of the invention is a diagnostic kit for the diagnosis of inflammatory disorders, preferably rheumatoid arthritis, comprising an antibody.
Another object of the invention is a diagnostic kit for the diagnosis of inflammatory disorders, preferably rheumatoid arthritis, comprising a polynucleotide of SEQ ID NO 9.